Amplification of the in situ hybridization signal by silver postintensification: the biotin-dUTP-streptavidin-peroxidase diaminobenzidine-silver-gold detection system

Frozen and vibratome sections from the adrenal gland of the rat were hybridized in situ using a biotinylated oligonucleotide probe specific for tyrosine hydroxylase (TH) messenger ribonucleic acid (mRNA). Hybridization was detected using the streptavidin-peroxidase-diaminobenzidine (DAB) system in c...

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Bibliographic Details
Published inHistochemistry (Berlin) Vol. 96; no. 4; p. 339
Main Authors Liposits, Z, Petersen, S L, Paull, W K
Format Journal Article
LanguageEnglish
Published Germany 01.08.1991
Subjects
3,3'-Diaminobenzidine
Adrenal Medulla - chemistry
Adrenal Medulla - enzymology
Adrenal Medulla - ultrastructure
Animals
Bacterial Proteins
Base Sequence
Chromaffin Granules - chemistry
Cytoplasm - chemistry
Gold
Histocytochemistry
Male
Microscopy, Electron
Molecular Sequence Data
Nucleic Acid Hybridization
Peroxidase
Rats
Rats, Inbred Strains
Ribosomes - chemistry
RNA Probes
RNA, Messenger - analysis
RNA, Messenger - genetics
Silver
Streptavidin
Tyrosine 3-Monooxygenase - analysis
Tyrosine 3-Monooxygenase - genetics
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Summary:Frozen and vibratome sections from the adrenal gland of the rat were hybridized in situ using a biotinylated oligonucleotide probe specific for tyrosine hydroxylase (TH) messenger ribonucleic acid (mRNA). Hybridization was detected using the streptavidin-peroxidase-diaminobenzidine (DAB) system in combination with silver-gold postintensification. The signal appeared as a black coloration and was localized to the cytoplasm of catecholamine-synthesizing chromaffin cells in the adrenal medulla. This coloration was due to the deposition of the silver-gold intensified DAB chromogen onto the probe hybridized to mRNA in carrier organelles. Compared with the conventional peroxidase-DAB labelling, the silver-gold amplified version was more sensitive in detecting TH mRNA. Using this modification, we were able to adapt the procedure to electron microscopy, thereby further localizing the hybridized signal to ribosomes. Because this hybridization detection system produces grains, not just color, this method has the potential for measurement of changes in mRNA levels at the ultrastructural level.
ISSN:0301-5564
DOI:10.1007/BF00271355